Caveolae belong to a class of membrane specializations called lipid domains. The key functions of caveolae are to internalize molecules for delivery to the cell interior and to compartmentalize the molecular machinery for specific signaling events that occur at the cell surface. This grant proposal outlines four projects that focus on achieving a molecular understanding of how caveolae capture signaling molecules, internalize them and then move to various locations in the cell. Aim #1 is to identify molecular addresses responsible for targeting signaling molecules to caveolae. This project focuses on the EGF receptor (EGFR), which is a growth factor receptor that is highly enriched in caveolae. The goal is to determine how EGFR is targeted to caveolae. Aim #2 outlines experiments to identify the molecules that control caveolae invagination and budding. An in vitro budding assay, which was developed previously in this laboratory to study coated pit budding, will be used to identify and characterize critical regulatory and structural molecules that mediate caveolae budding. Aim #3, which compliments the second aim, is to understand the molecular basis of caveolae vesicle traffic. Caveolin- GFP will be used to monitor caveolae membrane traffic when candidate regulatory proteins are reduced or eliminated from the cell using small interfering RNAs. Finally, a project to study signal transduction from caveolae is planned. This project has three goals. The first is to determine if EGFRs send specific signals only when they are in caveolae (site-specific signal transduction). Second, use proteomics to study and characterize molecular connections that are established between signaling molecules in caveolae when cells are stimulated in various ways. Finally, use new in rive techniques that have been established in the laboratory to target sensor molecules to caveolae and study signaling activities such as tyrosine phosphorylation, activation of Ras or calcium entry as they occur in living cells. Many signaling molecules are targeted to caveolae, including mutant forms that signal aberrantly from this location. Bacterial and viral pathogens have been found to commandeer caveolae to enter the cell. The data from this project may lead to the development of new strategies in treating human diseases.